MRS Muscle Function Biomechanics PDF

NMR IN BIOMEDICINE NMR Biomed. 2006; 19: 927953 Published online in Wiley InterScience (www.interscience.wiley.com) DOI:10.1002/nbm.
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Dynamic MRS and MRI of skeletal muscle function and biomechanicsy

Jeanine J. Prompers,1 Jeroen A. L. Jeneson,1 Maarten R. Drost,2 Cees C. W. Oomens,3 Gustav J. Strijkers1 and Klaas Nicolay1*
1 2
Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands Department of Movement Sciences, Maastricht University, Maastricht, The Netherlands 3 Laboratory for Biomechanics and Tissue Engineering, Department of Biomedical Engineering, Eindhoven University of Technology, The Netherlands Received 22 February 2006; Revised 21 July 2006; Accepted 22 July 2006
ABSTRACT: MR is a powerful technique for studying the biomechanical and functional properties of skeletal muscle in vivo in health and disease. This review focuses on 31P, 1H and 13C MR spectroscopy for assessment of the dynamics of muscle metabolism and on dynamic 1H MRI methods for non-invasive measurement of the biomechanical and functional properties of skeletal muscle. The information thus obtained ranges from the microscopic level of the metabolism of the myocyte to the macroscopic level of the contractile function of muscle complexes. The MR technology presented plays a vital role in achieving a better understanding of many basic aspects of muscle function, including the regulation of mitochondrial activity and the intricate interplay between muscle ber organization and contractile function. In addition, these tools are increasingly being employed to establish novel diagnostic procedures as well as to monitor the effects of therapeutic and lifestyle interventions for muscle disorders that have an increasing impact in modern society. Copyright # 2006 John Wiley & Sons, Ltd. KEYWORDS: energy metabolism; mitochondrial function; transgenic mice; diabetes; diffusion tensor imaging; ber tractography; elastography; tagging; velocity encoding
INTRODUCTION
There is an increasing need for advanced non-invasive technologies to assess tissue function and metabolism in vivo. Such technologies allow for determination of vital tissue properties in the normal state, but also enable the evaluation of dynamic changes in tissue status under diseased conditions. A number of human diseases that primarily affect skeletal muscle have a rapidly increasing socioeconomic impact, not only among the elderly (e.g. the development of pressure sores that develop as a consequence of prolonged mechanical loading of skeletal muscle and affect a remarkably high percentage of patients in hospitals) but also among young people (e.g. type 2 diabetes, which is rapidly reaching epidemic proportions around the world). The improved clinical management of such disorders critically depends on the development of innovative assays to support their diagnosis, to monitor the consequences of interventions and, perhaps most importantly, to establish objective
indices that can be used to prevent these diseases from developing in the rst place.
Why MRS and MRI?

Muscle function critically depends on a highly nonequilibrium ATP/ADP concentration ratio in the cytosol driving cyclic myocyte contraction and relaxation. These events are brought about at the molecular level by the actomyosin- and calcium-ATPases of the myolaments and the sarcoplasmic reticulum respectively. In skeletal muscle, three major sources of ATP are available to maintain a high cytosolic ATP/ADP ratiomitochondrial oxidative phosphorylation, glycolysis and creatine and adenylate kinase activity. MRS techniques are uniquely suited to quantitative measurement of each of these ATPgenerating activities in vivo. Furthermore, intramuscular storage and turnover of important nutrients, notably glycogen and lipids, can be monitored non-invasively by MRS. Alternatively, understanding of the basic principles governing muscle function, both in health and disease, requires knowledge of the tissue architecture at the level of the ber structure as well as insights into the viscoelastic properties of muscle tissue. A range of MRI techniques that have been recently introduced,
NMR Biomed. 2006; 19: 927953
*Correspondence to: K. Nicolay, Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. E-mail: k.nicolay@tue.nl y This paper is published as part of a Special Issue entitled NMR of the Musculoskeletal System. Copyright # 2006 John Wiley & Sons, Ltd.
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notably diffusion tensor imaging and MR elastography, report on these fundamental muscle properties. The use of proper signal preparation techniques allows the quantitative three-dimensional mapping of the above properties at the spatial resolution of regular macroscopic MRI. In a similar fashion, spatial encoding techniques can be employed for non-invasive quantication of the contractile function of the entire muscle by measuring the local displacement of the tissue resulting from contraction. The present review describes the basic principles as well as the major applications of dynamic MR spectroscopy (MRS) and imaging (MRI) techniques for measuring functional and biomechanical properties of skeletal muscle. A case will be made that MRI and MRS make important contributions towards improving the understanding of the aetiology as well as the development of effective therapeutic procedures for skeletal muscle disorders.
MR SPECTROSCOPY OF SKELETAL MUSCLE

MR spectroscopy has been widely used to study skeletal muscle metabolism. It enables a unique outlook on the in situ dynamics of tissue biochemistry by measuring the concentrations and/or turnover rates of metabolites [as reviewed earlier (14)]. This is primarily due to the noninvasive nature of the technique which allows serial timecourse measurements under conditions in which net changes in metabolite levels occur. This section will review classic work in the 1980s and 1990s and focus on recent innovative use of MRS to study dynamic aspects of muscle biochemistry in relation to function.
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Figure 1. Time series of dynamic 31P MR spectra of human quadriceps muscle at rest, during knee exion exercise and subsequent recovery (time resolution 28 s). The subject performed 2.5 min of single-legged knee exion exercise at 70% MVC. Four scans were averaged per spectrum (time resolution 7 s). The rst spectrum was obtained at rest. The last ve spectra were recorded during recovery. (Courtesy of J. J. Prompers et al., Eindhoven University of Technology)
P MRS
One of the best-studied and most insightful demonstrations of the generic power of MRS in the non-invasive investigation of metabolism in living mammalian tissues has been the use of 31P MRS to measure, in skeletal muscle, the response of ATP metabolism to exercise and subsequent recovery. 31P MR spectra of skeletal muscle typically show ve major resonances from inorganic phosphate (Pi), phosphocreatine (PCr) and adenosine triphosphate (ATP) respectively (Fig. 1). In addition, three more pieces of information that are of functional relevance can be retrieved from the 31P spectra, albeit indirectly, i.e. the intracellular pH, the free concentration of adenosine diphosphate (ADP) and Mg2 ions. Tissue pH can be deduced from the chemical shift of Pi. The 31P MR pH measurement makes use of the fact that the basic and acidic species of Pi that coexist at physiological pH have different chemical shifts and are in rapid chemical exchange (5). The relative proportion of the two species, i.e. pH, determines the actual resonance position. Following proper calibration, the measured Pi resonance frequency can thus be used to estimate the intracellular pH. Secondly, the CK equilibrium can be used to calculate
Copyright # 2006 John Wiley & Sons, Ltd.
the free levels of ADP in the cell (6). The concentration of ADP in resting and moderately active muscle is typically in the tens of micromoles range, which is too low to allow direct detection by 31P MRS. Knowledge of ADP levels is highly relevant, since ADP is an important regulator of the mitochondrial ATP synthesis activity, the main energy-delivering process in mammalian cells. This use of the CK reaction is based on the fact that total CK activity exceeds the ATP utilizing and delivering uxes several fold, implying that dynamic changes in PCr levels can be used indirectly to quantify changes in the ADP (and ATP) levels. Figure 2 shows an example of the kinetics of PCr recovery following exercise and the ADP recovery, as calculated from the CK equilibrium. Finally, the concentration of free Mg2 in the cell is of considerable interest since the Mg2 complexes of ADP and ATP are the active substrates for ATPases and kinases. This important variable can be deduced from the chemical shift separation between the a- and bor g-ATP resonances (7,8). Quantitative analysis of 31P MRS measurements of muscle during and after exercise. 31P MRS has opened a window on bioenergetics during skeletal muscle exercise and recovery in a non-invasive manner and with a time resolution typically of the order of seconds. This has made a major contribution to the understanding of mammalian cell energy metabolism, its control and the way in which it can be affected in disease. Figure 1 shows a typical time series of 31P MR spectra of human muscle acquired serially at rest, during exercise and subsequent recovery. At the start of the energy challenge, hydrolysed ATP is resynthesized from PCr breakdown and,
NMR Biomed. 2006; 19: 927953 DOI: 10.1002/nbm
SKELETAL MUSCLE BIOMECHANICS AND FUNCTION
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Figure 2. PCr (a) and ADP (b) recovery curves for an individual human subject as deduced from dynamic 31P MRS. Monoexponential curves (dark lines) were tted to the actual data (lled circles), which were obtained with a time resolution of 6 s. The time constants for PCr and ADP recovery were 26.9 and 11.8 s respectively. (Courtesy of J. J. Prompers et al., Eindhoven University of Technology)
depending on conditions, from anaerobic glycogenolysis. Thus, PCr levels decrease and Pi levels increase, while ATP levels remain constant (Fig. 1). PCr hydrolysis consumes protons and generally results in a small rise in cellular pH (alkalinization) at the start of exercise. During aerobic exercise, most of the energy is subsequently provided by oxidative metabolism. Anaerobic glycogenolysis produces protons via lactate production and typically results in cellular acidication. After the energy challenge, the PCr buffer is restored and Pi levels normalize (Fig. 1). The ATP used for resynthesis of PCr is mostly derived from oxidative phosphorylation. Several approaches to quantitative analysis and interpretation of 31P MRS measurements of energy balance in muscle during and after several types of exercise have been proposed [reviewed by Kemp and Radda (9)]. It is thus possible to estimate (a) the rates of glycogenolytic and aerobic ATP synthesis, (b) the oxidative capacity, (c) the proton efux and (d) the buffer capacity, as will be reviewed below. The distinction between various types of exercise previously made by Kemp and Radda (9), i.e. ischemic exercise, pure aerobic exercise under steady-state condition or during work jumps and mixed exercise, has been followed here. Glycogenolytic and aerobic ATP synthesis. During ischemic exercise, both proton efux and oxidative ATP synthesis are negligible. Therefore, the glycogenolytic ATP production can be directly calculated from changes in pH, corrected for the number of protons consumed by PCr hydrolysis calculated from changes in the PCr concentration and the rate at which the cell buffers protons (when the buffer capacity is known). Conley et al. studied the regulation of glycolysis using 31P MRS during ischemic stimulation of the human forearm (10). They showed that the glycolytic rate is proportional
to the muscle stimulation frequency but does not depend on metabolite levels and intracellular pH, which would be consistent with dominant control of glycolysis by factors other than ATP hydrolysis products that scale with nerve ring frequency (e.g. the free calcium concentration). It is important to note that the use of 31P MRS for quantifying glycogenolytic activity only holds under ischemic conditions. This has been elegantly shown by Hsu and Dawson (11) in comparative 1H and 31P MRS studies on excised frog muscle. During pure aerobic exercise under steady-state conditions, oxidative ATP synthesis can be derived from measurements of (a) oxygen consumption, (b) the ATPase exchange rate determined by magnetization transfer (performed during steady-state exercise; see below) or (c) the mechanical work rate. Specically, with regard to the latter, the methodology developed by Chance involves 31P MRS measurements of energy balance at multiple workloads in a ramp protocol (12) (Fig. 3). From such a dataset, the kinetic transfer function of poweroutput and an index of the cytosolic free ADP concentration can be derived, yielding an estimate of the maximal mitochondrial ATP synthesis ux of the muscle (12,13). Accuracy of this particular approach, however, critically depends on quantication of contractile work on the one hand and restriction of 31P MRS signal collection to the active muscle group(s) recruited during the exercise (14). As such, it is less robust with respect to experimental design than determination of PCr recovery rate following mild exercise (see below). This complication in experimental design employing voluntary muscle contraction has, however, been successfully overcome by electrical stimulation of muscle contraction (1517). During pure aerobic work jumps, dynamic measurements of the initial rate of PCr depletion yield an estimate of the total rate of ATP synthesis (Fig. 4).
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Figure 3. Time series of steady-state 31P MR spectra of the medial head of the quadriceps upper leg muscle of a healthy male subject performing a bicycling exercise at incremental workloads (in W) to exhaustion in a ramp protocol. Each workload was maintained for 5 min. Pedaling frequency was 1 Hz. For each MR spectrum, 180 scans were averaged during minutes 3 to 5 at each workload (i.e. after ATP metabolism had reached a new steady state). Prior to Fourier transformation, a 10 Hz line broadening was applied. [Modied with permission from (79)]
Typically, at low workloads, glycogenolysis can be neglected. During mixed exercise, however, lactic acid production and therefore proton efux can be signicant. At the start of exercise, when the pH has not dropped signicantly, proton efux can be neglected and the initial rate of glycogenolytic ATP synthesis can be calculated as described for ischemic exercise. Later during exercise, when the pH has fallen substantially, the glycogenolytic ATP synthesis rate can be estimated from the proton production rate, corrected for proton efux, if the contribution of oxidative ATP synthesis is ignored. The proton efux could be calculated from recovery (see below). More ambitiously, both glycogenolytic and oxidative ATP synthesis rates can be estimated by a calculation that uses total proton production and total ATP turnover (Fig. 4). In this approach, the total ATP turnover can be determined in different ways, i.e. (a) by calibration from ischemic exercise at the same power, (b) from the non-oxidative ATP synthesis rate in the rst exercise interval, when the oxidative ATP production is still small or (c) from very early changes in the PCr concentration alone (neglecting both glycogenolytic and oxidative ATP synthesis). In a recent study, Lanza et al. investigated whether age affects ATP synthesis rates from the different pathways during maximal voluntary isometric contractions (18). Oxidative ATP synthesis rates were similar in young and older men, but anaerobic glycolytic ATP synthesis rates were higher in young men, suggesting that the relative proportion of oxidative ATP production increases with age.
Figure 4. Energy balance [(A) and (B)] and proton balance (C) in human forearm muscle computed from 31P MRS data of ATP, PCr and Pi peak intensities and chemical shifts during electrical stimulation of the ulnar and radial nerve at 0.6 Hz (o) and 1.6 Hz (&). Solid lines in (A) and (B) represent ts of Meyers monoexponential relation to the PCr/(PCr Pi)(t) data for each stimulation frequency. The dashed line in (A) represents the derivative of the PCr time course at t 0 during serial nerve stimulation at 1.6 Hz and equals the ATP consumption rate at this particular contraction frequency. The dashed lines in (C) represent ts of linear functions to the pH(t) data for each stimulation frequency and are a measure !lac of the non-oxidative ATP synthesis ux (JGly ). [Modied p with permission from (16)]
Energetic recovery from exercise is mostly a function of mitochondrial ATP synthesis and can therefore be regarded as an aerobic work jump (19). As such, oxidative ATP synthesis can be estimated from the absolute rate of PCr resynthesis during recovery plus a small correction for basal ATP turnover. Oxidative capacity. The oxidative capacity or Qmax of the mitochondrial pool in a muscle is a function of the density and capacity of working mitochondria and the supply of substrate and oxygen, but is independent of
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muscle mass. Therefore, it has proven to be a valuable parameter to evaluate muscle function. Qmax can be inferred from steady-state exercise, work jumps or recovery, as will be described below. For pure aerobic exercise at steady state, quantitative mechanistic relationships between the oxidative ATP synthesis (derived from oxygen consumption, the ATPase rate or the mechanical work rate) and possible cytosolic effectors have allowed for estimation of mitochondrial oxidative capacity (20). The two approaches that have been most widely applied since their formulation in the mid-1980s are both mechanistically based in feedback control of mitochondrial respiration involving a function of adenine nucleotide concentrations: (a) the kinetic control model involving ADP stimulation of respiration (21) and (b) the non-equilibrium thermodynamic control model involving a linear dependence of respiration on the free energy of ATP hydrolysis (DGATP) (22). In the kinetic control model, the mitochondrial oxidative ATP synthesis rate Q has been generally described by a hyperbolic dependence on the ADP concentration ([ADP]), characterized by a maximum rate Qmax and an apparent ADP afnity K50 (21) 1 Q Qmax K50 1 ADP (1)
P MRS data. Firstly, it was shown that ADP stimulation of respiration follows apparent cooperative instead of MichaelisMenten kinetics (25). Secondly, careful analysis of complete dynamic 31P MRS datasets of restcontractionrecovery experiments showed that a feedforward respiratory control mechanism, likely involving calcium and similar to that found in cardiac muscle (26), may be additionally activated during contractile work (27). Equations (1) and (2) should be revised accordingly to encompass these new insights. The linear steady-state relationships described above imply exponential kinetics during pure aerobic work jumps with a rate constant kT equal to the steady-state proportionality constant: dQ Qmax (3) dPCr tCr Thus, Qmax could be estimated by multiplying the rate constant, obtained from kinetic measurements of the exponential change in [PCr], by the [tCr] or (almost equivalently) the resting [PCr]. The linear model predicts that the rate constant is independent of the power level, and this was indeed shown experimentally (22,28). Qmax can also be derived from measurements during recovery from exercise, in a similar way to that described for aerobic exercise. Multiple pairs of initial PCr resynthesis rates and ADP concentrations can be analyzed by means of a Hanes plot to determine Qmax and K50 according to the kinetic control model. More exactly, the Hanes plot analysis can be modied to incorporate the basal rate of ATP synthesis. In the linear approximation of the ADP control model, Qmax can be estimated as the product of the rate constant of PCr recovery and the resting [PCr], as for aerobic work jumps. Alternatively, in many studies, the PCr recovery rate constant (or time constant or half-time) by itself (Fig. 2A) has been used as a measure for mitochondrial function (2932). However, this might lead to misinterpretations if the end-exercise [PCr] and [ADP] differ signicantly between the subjects studied. In this case, the PCr recovery rate constant might differ while Qmax is still the same. Furthermore, it has been shown that cytosolic pH has a strong inuence on the kinetics of PCr recovery (3,29,33,34). Therefore, in the case of low end-exercise pH, the PCr recovery rate constant is not a good measure of mitochondrial function. Under these particular conditions, the kinetics of ADP recovery (Fig. 2B), which is independent of end-exercise pH, provides a robust index of mitochondrial function (29,30,35). Finally, using sophisticated statistical analysis of PCr recovery data, Meyer has found that multiple velocities of ATP supply contribute to energetic recovery in mildly stimulated muscle of mixed ber type (19). Moreover, during high workloads, the analysis uncovered a rapidly decaying (time constant < 10 s) extra ATP synthesis capacity that was not activated during mild stimulation and possibly reected extra mitochondrial [e.g. as a result of feedforward respiratory control kT
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Qmax can be estimated from a single-point analysis by assuming some value for K50. However, K50 is a variable that may differ between cell types (23). When steadystate exercise is performed at several power outputs, Qmax as well as K50 can be determined from a multipoint analysis using non-linear curve tting or, in the absence of sophisticated computations, on the basis of Lineweaver Burk or Hanes linearization plots. The non-equilibrium thermodynamic model holds that respiration varies linearly with the thermodynamic driving force DGATP during submaximal work (22). It was later shown that the precise relation is quasi-linear (13). The metabolic restraints imposed by the creatine kinase equilibrium make it impossible to distinguish the nonequilibrium thermodynamic control model from the kinetic control model when pH changes are small (20,24). In that particular case, [ADP] can be approximated by the [Pi]/[PCr] ratio to yield a relation in terms of concentrations that are directly measurable by 31P MRS (21). In an alternative linear formulation of the kinetic control model, equation (1) reduces to PCr (2) Q % Qmax 1 tCr where [tCr] is the total creatine concentration. This formulation is equivalent to the non-equilibrium thermodynamic control model. Two corrections of the mitochondrial respiratory control model have more recently allowed for improved accuracy of the estimation of mitochondrial Qmax from
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(15,16)] or simply anaerobic glycogenolytic uxes. Therefore, more sophisticated analysis of 31P MRS PCr recovery data than simply tting a monoexponential to the data is generally recommended. Proton efux. During recovery from exercise, cytosolic pH will recover to the resting value, and this process is a function of net proton efux (36). The change in proton concentration in the cell can be calculated from the change in pH multiplied by the cytosolic buffer capacity and equals the proton efux rate minus the rate of proton generation by PCr resynthesis and aerobic ATP production. Proton efux during recovery is pH dependent, with values from 10 mmol/L/min around pH 6 to near-zero around pH 7 (37). Several mechanisms are responsible for proton efux, such as sodium/ proton exchange, sodium-dependent chloride/bicarbonate exchange, efux of undissociated lactic acid, and outward proton/lactate cotransport. Cea et al. investigated skeletal muscle metabolism in patients with dermatomyositis and polymyositis (38). In both groups of patients, PCr and ADP recovery half-times were almost twice as long as in controls, suggesting decreased mitochondrial function. However, the rate of proton efux was also signicantly reduced, indicating that the impaired muscle function is more likely to be secondary to an impaired blood supply, rather than an intrinsic mitochondrial defect. Buffer capacity. The quantitative measurement of proton efux and glycogenolytic ATP production from 31 P MRS data relies on knowledge of the cytosolic buffer capacity. The buffer capacity of a muscle cell is typically 3050 slykes and is composed of three components (9,37): (a) Pi (up to 10 slykes in exercise), (b) bicarbonate (less than 5 slykes in muscle that is effectively closed to carbon dioxide) and (c) a nonPi/non-bicarbonate component, mainly from carnosine and imidazole groups in histidine residues in proteins. The latter component can vary from about 16 slykes at pH 7 to about 38 slykes at pH 6. Cytosolic buffer capacity can be estimated as the ratio of the change in proton concentration and the change in pH from measurements during exercise and recovery, as will be described below. During ischemic exercise, both proton efux and oxidative ATP synthesis can be ignored. Therefore, the buffer capacity can be calculated from changes in pH, [PCr] and [lactate]. It can be shown that, in the case of ischemic exercise at constant power, the buffer capacity can be calculated from changes in pH and [PCr] alone (37). At the start of aerobic exercise, much of the change in proton concentration is due to changes in [PCr], as glycogenolysis, oxidative ATP production and proton efux are still negligible. Therefore, the buffer capacity can be approximated from very early changes in pH and [PCr] alone. The validity of this approach can be
questioned, as it is known that glycogenolysis is substantially activated even before the pH starts to decrease. During recovery from exercise, lactate production is zero. However, to calculate the buffer capacity from changes in pH and [PCr] at the start of recovery, a correction is necessary for proton efux. To this end, the mean rate of pH recovery during the later near-linear phase of pH recovery can be extrapolated to the start of recovery to calculate a corrected end-exercise pH. This approach gives good estimates for the buffer capacity when the end-exercise pH is not very low. Using this method, Newcomer et al. determined the non-Pi/nonbicarbonate buffer capacity of the human gastrocnemius/ soleus muscle groups using 0.5 s time-resolved 31P MRS at 4.1 T (39). Spectroscopic imaging studies. The vast majority of 31 P MRS studies of muscle energetics reviewed thus far, including the pioneering report by Ackerman et al. in Nature (40), employed localization of a tissue volume of interest by physically placing a customized RF receiver probe (surface coil) of desired dimensions directly over the appropriate body part. In this way, use of a simple pulse-acquire sequence in combination with the particular sensitivity prole of the probe ensured that the majority of the detected lumped MR signal originated from the volume of interest. 31P MRS studies of human limb muscles have typically employed this strategy with great success. Conversely, a number of MRS acquisition techniques employing some form of controlled magnetic eld gradient manipulation [i.e. of either B0 (41,42) or B1 (43)] to spatially encode spins were developed in the late 1980s/early 1990s that allow for 31P MRS measurement of ATP, PCr, Pi and H concentrations in deeper-lying tissue [e.g. muscle (4347), heart (4850), brain (5154) and intestinal organs (50,55)]. With appropriate spatial resolution, these techniques have even allowed for investigation of spatial energetic heterogeneity within a particular organ itself [e.g. (45)]. Although these early studies demonstrated the potential of the method, these particular 31P MR spectroscopic techniques have since been seldomly used. This has been primarily because the intrinsically low MR sensitivity of the 31P nucleus curbed the spatial resolution that could be achieved at eld strengths available at that time (1.5 2 T) within a normal patient exam time. In muscle, this shortcoming precluded any application to dynamic studies of energy metabolism, restricting investigations typically to stationary metabolic states [but see (56)]. Two recent developments warrant a revival of this 31P MRS method. Firstly, modern human MR magnets nowadays operate at much higher eld strengths, with 3T and 7T rapidly becoming the clinical versus research standard, respectively. Secondly, signicant progress has been made in SNR enhancement strategies for MR imaging of low-g nuclei such as 31P, yielding improved
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temporal and spatial resolution of spatially localized spectroscopic techniques (57,58). At the same time, the spread of diabetes type 2 through modern Western societies has been reaching epidemic proportions requiring imaging modalities capable of probing both vascular function as well as its impact downstream on muscular oxygen and energy metabolism in humans. Elegant studies by Richardson et al. (59) and Greenman (57) have demonstrated the high potential of and need for combined 1 H MRI and modern spatially localized 31P spectroscopic techniques in this particular eld of clinical investigation. Metabolite exchange kinetics under steady-state conditions. A very elegant feature of MRS is that under certain conditions the measured signal strength can be sensitized to the rate of metabolite turnover even under steady-state conditions, i.e. in the absence of net changes in metabolite levels. There are two possible ways to do this. The rst measurement mode makes use of magnetization transfer between nuclei that are linked by chemical exchange, while the second version of this steady-state kinetics measurement uses isotope-enriched substrates. Transfer of magnetization can be studied by the selective perturbation of the equilibrium magnetization of one of the nuclei that is involved in the exchange process and measuring the effect on the strength of the signal from its exchange partner (6062). One of the best-studied examples of MT concerns the enzyme creatine kinase (CK), which is abundantly present in skeletal muscle (63). CK catalyzes a phosphoryl exchange reaction, in which a phosphate moiety is transferred from the g-position of ATP to PCr, and vice versa. As described above, the CK reaction is of considerable functional relevance, since PCr acts as an energy buffering substance. The mathematics of chemical exchange is well worked out for MR, and this enables the quantication of the exchange process in terms of the ux rates. The MT experiment can be done in several ways. A popular procedure involves the complete saturation of one of the resonances involved in the exchange with a long frequency- selective RF pulse and the quantication of the reduction in the signal intensity of the exchange partner (62,64). This saturation transfer experiment is routinely conducted by saturating the g-ATP resonance and measuring the reduction in PCr peak intensity (21,65). Quantication of CK kinetics in terms of the unidirectional rate constants of the forward and reverse reactions requires additional knowledge of the longitudinal relaxation times T1 of the nuclei involved in the transfer process, in the absence of exchange. Measurements of CK ux in skeletal muscle as a function of workload have shown that CK kinetics is rather insensitive to alterations in ATP demand and greatly exceeds maximal ATP turnover rates (66,67). The latter is a prerequisite for the use of the CK equilibrium for the estimation of free ADP levels in the cell.
P saturation transfer has also been utilized to measure the kinetics of the mitochondrial ATP-synthase in rodent and human skeletal muscle, under resting conditions (6872). This experiment was conducted by selective saturation of the g-ATP resonance and quantication of the reduction in the intensity of the Pi signal (6871). Shulman and coworkers have employed this technology to study mitochondrial functionality in elderly patients (73) as well as in insulin-resistant offspring of type 2 diabetic parents (74,75). The latter studies provided evidence of impaired mitrochondrial functionality and contribute to the understanding of the aetiology of insulin resistance and type 2 diabetes. Padeld et al. (72) have used a similar method to assess the effects of burn injury on mitochondrial activity in mouse skeletal muscle. While having only minor effects on the 31P MR spectra as such, burn injury appeared signicantly to reduce the rate of mitochondrial ATP synthesis.
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P MRS of metabolite diffusion. A number of 31P MR spectroscopy studies have examined the diffusive properties of low molecular weight metabolites in skeletal muscle (7678). These studies were inspired by the notion that diffusional displacements of high-energy phosphates are required to connect the energy-requiring and energydelivering sites within the muscle cell. Knowledge of these diffusivities thus provides insights into the maximal diffusion-based high-energy phosphate uxes. The data indicate that the in vivo apparent diffusion coefcient (ADC) of both ATP and PCr amount to circa 90% of their in vitro values at 378C. By measuring the metabolite ADCs as a function of the diffusion encoding time, the cross-sectional dimensions of the cylindrical restriction compartment were estimated to be circa 20 mm in rat hindleg muscle (78), which is considerably smaller than the values obtained by microscopy (6080 mm). This remarkable nding suggests that in vivo the restriction of ATP and PCr diffusion primarily results from intracellular structures and not from the myocyte cell membrane. The above metabolites are essentially trapped inside the cell and therefore allow probing of the diffusive characteristics of the intracellular compartment. Measurements of water diffusion do not necessarily offer this opportunity, as water readily exchanges between intra- and extracellular compartments (see the section below on MR imaging of skeletal muscle).
Novel insights into muscle biology learned from P MRS studies. A rst set of important lessons learned has been related to the hitherto unprecedented non-invasive nature of the method. In the early pioneering days of the application, 31P MRS measurements showed that the absolute Gibbs energy of ATP hydrolysis in resting muscle was some 20 kJ/mol higher than classically thought on the basis of biochemical analysis of freeze-clamp samples of tissue, i.e. 65 kJ/mol instead of 45 kJ/mol respectively. This important result
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conrmed predictions made by Lawson and Veech on the basis of enzyme equilibrium calculations (6). Analogously, 31P MRS observations on proton balance in exercising human quadriceps muscle engaged in exhaustive natural (i.e. two-legged) locomotion activity recently showed that muscle cell pH does not drop nearly as muchi.e. maximally 0.2 instead of 0.5 pH unitsas thought on the basis of classic biochemical analysis of freeze-clamp samples of tissue (79). A second set of lessons in muscle biology relates to the eld of metabolic control, in particular the in vivo kinetic regulation of mitochondrial ATP output. Observations by 31 P MRS in resting and actively contracting muscle conrmed Chances early prediction of a primary role of ADP stimulation of respiration, culminating in accurate characterization of the overall kinetic transfer function of oxidative phosphorylation (25) (for a more detailed review of this particular literature, the reader is referred to the contribution by Chance in this volume). A third set of novel insights has come from application of 31P MRS to the investigation of muscle cell energetics in selected transgenic murine models of the ATP metabolic network in mammalian cells, and is the focus of this part of the review. 31P MRS studies of the CK isoenzyme system (63) as well as the closely linked adenylate kinase (AK) isoenzyme family (80) in skeletal muscle have received an enormous boost with the generation of knock-out mice, in which one or more members of the CK or AK isoenzyme family were completely eliminated. 31P MRS has played a critical role in establishing the functional consequences of the loss of enzyme function. van Deursen et al. (81) were the rst to show that mouse skeletal muscle decient in the (most abundant) M-type CK isoenzyme lacks burst activity and has improved endurance properties compared with wildtype muscle. In spite of the formidable decrease in the total CK activity in the knock-out muscle, the remaining levels of the B- and mitochondrial CK isoenzymes sufced to support similar rates and extents of PCr decline with exercise as those observed in wild-type muscle (81). Saturation transfer 31P MRS studies of M-CK / muscle led to a negligible transfer of magnetization from the PCr to the g-ATP resonance (81,82). A similar effect is illustrated in Fig. 5 for skeletal muscle in a mouse that is decient in both the M- and mitochondrial CK isoenzyme (83,84). One of the surprising ndings of the 31P MRS studies in this CK- decient mouse is the presence of PCr in the spectrum, which was explained by the (low level of) residual B-type CK remaining in the knock-out tissue. van Deursen et al. (85) have performed very elegant 31P MRS saturation transfer studies in skeletal muscle of mice with a graded expression of the dominant M-type CK isoenzyme. The ux through the CK reaction remained detectable by means of 31P MRS only at relatively high levels of M-CK expression (i.e. above 34% of wild-type levels). The reason for this apparent non-linearity of the measured CK ux with CK enzyme activity remains to
Figure 5. Magnetization transfer 31P MRS of mouse skeletal muscle. Left, spectra obtained from the hindlimb of a wildtype mouse; right, spectra from a CK-decient mouse. (A) Control spectra collected during low-power selective irradiation on the low-eld side of the PCr peak; (B) spectra obtained with selective saturation of the g-ATP resonance; (A B) the difference spectra, which show a large effect of gATP saturation on the PCr peak in wild-type muscle and the absence of an effect for CK-decient muscle. [Reproduced with permission from (84)]
be elucidated. Interestingly, van Deursen et al. (85) observed that the ability of the skeletal muscle in the graded CK mutants to perform burst activity was also subnormal and closely correlated with the M-CK expression level. ter Veld et al. (86) provided evidence that CK deciency results in a distinct adaptive change in the muscle mitochondrial phenotype. This aids in rescuing myocellular energy homeostasis, in spite of the complete loss of the PCr/Cr energy buffering system. Recently, Roman and colleagues (87) have conducted detailed 31P MRS studies of the consequences of M-CK deciency on PCr depletion kinetics at the onset of contractions in skeletal muscle of M-CK / mice. The initial rate of PCr depletion was almost 5 times lower in the knock-out as compared with the wild-type muscle. A kinetic model of muscle energy metabolism suggested that cytoplasmic ADP more rapidly increased and mitochondrial oxidative phosphorylation was more rapidly activated at the onset of contractions in M-CK / compared with wild-type muscles. Heerschap and colleagues have also used 31P MRS to evaluate the biochemical effects of and the functional adaptations caused by the loss of CK (88,89) and AK function (90) in mouse skeletal muscle by monitoring the response of the muscle to ischemia, as a model of metabolic stress. Koretsky and coworkers (9194) have studied the role of specic CK isoenzymes in the energy metabolism of mouse skeletal muscle. Transgenic mice homozygous for muscle expression of the brain- type B-CK were studied by 31P MRS (93). The effects of electrical stimulation of the hindleg muscle complex were similar in transgenic and wild-type muscle, indicating that a 50% increase in CK activity has a negligible effect on skeletal muscle metabolism or contractile function. When replacing the muscle-type M-CK by the brain-type B-isoform
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(94), the aberrant contractile phenotypes seen in M-CK knock-out mice were normalized. These ndings suggest that there is functional equivalence among the CK isoforms in cellular energy metabolism. Recently, Hancock et al. (95) demonstrated that mouse skeletal muscle lacking the adenylate kinase isoenzyme 1 (AK1) shows a remarkably strong increase in the free ADP levels during fatiguing, repetitive contractions. ADP levels rose to circa 1.7 mM and thereby even became directly visible by 31P MRS (Fig. 6). Although these high ADP levels must have serious implications on cellular processes that depend on energy from ATP hydrolysis (e.g. Ca2 pumps), AK1 / and wild-type muscles exhibited similar rates of fatigue. Similar 31P MRS studies have been done in muscle decient in the enzyme guanidinoacetate methyltransferase (GAMT) (96,97), which is responsible for the synthesis of creatine. 31P MR spectra of hindleg muscle of the GAMT / mice showed no PCr signal and instead showed the signal for phosphorylated guanidinoacetate (PGua), the immediate precursor of Cr, which is not normally present. During ischaemia, PGua was metabolically active in GAMT / mice and decreased at a rate comparable with the decrease in PCr in WT mice. However, the recovery rate of PGua in GAMT / mice after ischaemia was reduced compared with PCr in WT mice. Saturation transfer measurements revealed no detectable ux from PGua to g-ATP, indicating a severe
reduction in CK ux. The GAMT mouse can be regarded a model of an inborn error of metabolism. A very elegant aspect of this model is that creatine feeding can be used largely to restore the functions of the PCr/creatine system in skeletal muscle (97). Gene and stem cell therapy. 31P MRS is expected to make important contributions to the assessment of the potential functional benets of novel gene and stem cell therapies of musculoskeletal disorders, including rare genetic diseases such as Duchenne muscular dystrophy and frequently encountered disorders such as peripheral vascular disease. 31P MRS may also serve as a noninvasive tool to report on the level of gene expression following gene therapy, as demonstrated by the pioneering example of the virally-mediated transfer of the arginine kinase gene in mouse skeletal muscle (98). Arginine kinase was active, as evidenced by the fact that a phosphoarginine resonance appeared, which could be simply distinguished from the PCr peak and, alike PCr, was reduced during muscle ischemia.
1
1
H MRS
H MRS also has a lot to offer to in vivo research on skeletal muscle. Applications include: (a) the quantication of intramyocellular lipids (IMCLs), which has
Figure 6. 31P MRS of hindlimb skeletal muscle in wild-type (left) and adenylate kinase isoenzyme-1 (AK-1) decient mouse (right). Spectra were collected over the course of 64 (2 per second) isometric tetanic contractions, as indicated. In both cases a large decrease in PCr and increase in Pi levels were observed. Remarkably, by approximately 40 contractions, ADP was clearly visible in AK-1decient muscle (see arrow), while no such effect was observed in WT muscle. [Reproduced with permission from (95)]
Copyright # 2006 John Wiley & Sons, Ltd. NMR Biomed. 2006; 19: 927953 DOI: 10.1002/nbm
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proven to be a powerful tool in exercise and nutrition research as well as studies of the aetiology of insulin resistance and type 2 diabetes (for a review, see the contribution by Boesch and Schick elsewhere in this issue); (b) the detection of lactate formation; (c) measurement of the total creatine content of muscle, which is especially relevant for bioenergetic studies; (d) the (indirect) measurement of muscle ber orientation from the residual dipolar coupling effects of low molecular weight metabolites; (e) studies of transfer of magnetization between free creatine/phosphocreatine and a motionally restricted proton pool; (f) the measurement of metabolite diffusion in the muscle cell; (g) the measurement of tissue (de)oxygenation, based on the 1H MRS signal of deoxymyoglobin. Below, a brief review will be given of those aspects of 1H MRS that focus on in situ metabolite dynamics in muscle. Lactate formation. Lactate is the end-product of anaerobic metabolism and a source of H and therefore plays an important role in muscle metabolism and pH regulation. A number of methods have been proposed for indirect calculation of changes in lactate levels in exercising muscle from 31P MRS data (see above). It is obvious that the direct measurement of lactate with 1H MRS would have many advantages. The 1H MRS detection of lactate in muscle, however, has several problems that are, among others, caused by: (a) overlapping lipid signals; (b) dipolar coupling effects (see below); (c) the effects of T1 and T2. The incorrect interpretation of any of these effects will result in inaccurate estimations of lactate levels. Pioneering work on the use of 1H MRS to detect lactate formation during exercise in human skeletal muscle in vivo has been carried out by Hetherington et al. (99) and Pan et al. (100). Dawson and coworkers have extensively studied lactate formation in excised muscle with the use of 1H MRS (11,101,102) and have shown that the lactate levels as measured by 1H MRS are in good agreement with those obtained by chemical analysis of tissue extracts (11). Several studies have addressed the use of spectral editing techniques to allow the specic detection of the doublet peak of the lactate methyl protons at 1.33 ppm in vivo. Thus, Carlier and colleagues have used double-quantum coherence editing to detect lactate in exercising human skeletal muscle (103,104). For the above reasons, however, a reliable and reproducible procedure for the unambiguous quantication of lactate levels in skeletal muscle with in vivo1H MRS is still unavailable. Residual dipolar coupling effects. Skeletal muscle is a highly ordered tissue, at basically any length scale, and this has a number of very important consequences for 1 H MRS spectra of muscle tissue [for a review, see (105)]. Geometric ordering at the macroscopic scale is responsible for the characteristic separation of the 1H MRS signals from intra- (IMCL) and extramyocellular (EMCL)
lipids, which forms the basis of the widespread use of 1H MRS in studies of intramyocellular fat storage (105). The IMCL/EMCL peak separation depends on the orientation of the muscle with respect to the magnetic eld and amounts to a maximum of circa 0.2 ppm when the muscle is parallel with the magnetic eld. This chemical shift difference has been shown to originate from bulk magnetic susceptibility effects that are due to the layered ordering of EMCL depots along the main muscle axis, while IMCL is organized in spherical droplets in the cytoplasm of the muscle cell (105). Interestingly, several low molecular weight metabolites including creatine/ phosphocreatine, taurine and lactate also exhibit spectral features that depend on the orientation of the muscle with respect to the direction of B0 (105). These metabolites show a signicant residual dipolar coupling, which is indicative of incomplete motional averaging of the dipolar interaction between nearby protons, e.g. the two CH2 protons within the methylene group of Cr and PCr. The origin of these effects is not completely understood. int Zandt et al. (106) have demonstrated that the Cr/PCr residual coupling effect is not due to interaction with the creatine kinase enzyme, since CKdecient muscle shows the same effect as wild-type muscle. A major obstacle towards the elucidation of the origin of the residual coupling phenomenon is the lack of a clear-cut in vitro system with which the effect can be modeled. It has been shown that the 1H MRS signals from creatine as well as phosphocreatine and lactate exhibit similar residual couplings in an in vitro suspension of magnetically ordered bicelles to those that they exhibit in skeletal muscle in vivo (107). This nding demonstrates that the (unspecic) interaction of the metabolites with anisotropically ordered structures sufces to induce the residual coupling effect. However, denite proof that this also explains the in vivo observations is still lacking. Vermathen et al. (108) have elegantly made use of the residual coupling effect to map the local ber orientation in human skeletal muscle on the basis of 1H spectroscopic imaging data. It is important to note that the effects of muscle orientation on the 1H MR spectra may have profound consequences for the echotime dependence of the signals of low molecular weight metabolites from muscle (105,109) and thus for the quantication of metabolite content, in particular when using relatively long echo times. Magnetization transfer studies. Magnetization transfer phenomena in skeletal muscle have also been investigated with 1H MRS. These studies were inspired by the remarkable nding in 1H MRS studies of rat brain by Leibfritz and colleagues (110,111) that off-resonance saturation leads to a strong reduction in the PCr/Cr proton signal. Subsequently, De Graaf et al. (112) demonstrated that other metabolites in rat brain, including glutamate/glutamine and lactate, also exhibit a signicant
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off-resonance MT effect. When studying mouse, rat and human skeletal muscle, Kruiskamp et al. (82,113115) observed a pronounced MT effect for PCr/Cr in skeletal muscle as well. Similar conclusions were reached in a more recent study on human skeletal muscle by Renema et al. (116). A possible explanation for this nding is that the MT experiment probes the exchange of PCr/Cr between a mobile and a restricted conguration, in which the molecules are strongly immobilized and, possibly, less available for metabolic conversion. Mathematical modeling showed that the metabolite MT effect could be explained with a two-site exchange model in which the restricted metabolite pool has a T2 of circa 10 ms. The model also suggested that the immobile pool amounts to circa 12% of the total PCr/Cr pool in muscle. Interestingly, experiments in mouse skeletal muscle indicated that the 1H MT effect on the PCr/Cr protons was identical in CK-decient and wild-type tissue (82), implying that the interaction between PCr/Cr and the CK enzyme is not responsible for the immobilization effect. The precise mechanism responsible for and the biological signicance of the pronounced (phospho)creatine MT effect in 1H MRS remain to be established. Metabolite diffusion. As described above, diffusion MRS enables the measurement of the temporal and directional dependence of metabolite diffusion in skeletal muscle. This technique yields the effective diffusivity of the metabolite of interest in the cytoplasm. The acquisition of diffusion-sensitized MR spectra as a function of diffusion time and diffusion direction also allows the estimation of the dimensions of the compartment that restricts the diffusion (117). Kruiskamp et al. (118) studied the diffusion of water and total creatine in mouse hindleg skeletal muscle with 1H MRS and that of phosphocreatine with 31P MRS. Measurements in wildtype mouse skeletal muscle were compared with those in creatine kinase decient muscle. Firstly, the unbounded in vivo diffusion coefcients of water, Cr and PCr, as estimated by modelling the data, were similar to those in aqueous solution at 37 8C, suggesting that the viscosity of the cytoplasm is similar to that of water. Secondly, the restriction radius, i.e. the diameter of the cylindrical compartment by which the diffusion of the metabolites is restricted, was considerably smaller than the diameter of the muscle cells in both rat and mouse hindleg for water and in particular for the metabolites. The latter is presumably caused by restriction effects that are imposed by intracellular structures (e.g. mitochondria and sarcoplasmic reticulum) and not just the sarcolemma. Finally, the apparent diffusivities of water, PCr and Cr were identical, within experimental error, between wild-type and CKdecient muscle. This indicates that the wellknown plastic adaptation of muscle to CK deciency (81,83) has no effect on the effective metabolite diffusivity in the cytoplasm. The reported increase in mitochondrial density in CK-decient muscle, especially
in the myobrillar compartment (83), possibly represents an adaptation that is aimed to overcome a deciency in diffusion-based energy transport capacity. Studies of tissue oxygenation. Cellular oxygen status is a critical factor for muscle function, and therefore knowledge of the intracellular partial pressure of oxygen pO2 is of great importance for studies of both normal and diseased skeletal muscle. A variety of methods have been used to assess local pO2, including oxygen electrodes and near-infrared spectroscopy. The main drawback of these measurements is that they are either invasive or very difcult to quantify. 1H MRS enables non-invasive, in vivo measurement of the local pO2 with the use of the 1H signal of deoxymyoglobin (Mb), which is present in muscle tissue only. This technique has been pioneered by Jue and coworkers in isolated myocardium (119,120) and by Leigh and coworkers (121) in skeletal muscle. The Mb method is based on the detection of the N-delta proton signal from the proximal histidine F8. The visibility of the histidyl proton is determined by the heme iron, which shifts the resonance circa 78 ppm downeld of the water line in the paramagnetic, deoxygenated state. Upon oxygenation, Mb becomes diamagnetic and the shifted signal of the histidine F8 proton disappears. For that reason, until recently, the Mb method has only been used to determine increased deoxy-Mb levels, i.e. in the case of decreased pO2, during exercise and/or induction of muscle hypoxia/ ischemia by cuff occlusion, since these conditions are associated with increased Mb signal. In 1992, Kreutzer et al. (122) proposed a very interesting technique for measuring tissue pO2 that was based on the combined use of the signal from the F8 histidine proton in Mb (which shows increased signal upon deoxygenation) and the signal from the valine E11 g-methyl protons, which rather shows a maximal signal in the oxygenated state. The concept was demonstrated in perfused rat heart but has never seen a follow-up, neither in cardiac nor in skeletal muscle. The Mb method based on the histidyl F8 proton has been used for detailed studies of exercise physiology (59,123126) and de- and reoxygenation following venous occlusion and reperfusion (104,124), as well as the consequences of peripheral arterial disease (127). Carlier and coworkers have recently introduced a very powerful MR protocol, in which interleaved 1H and 31P MRS data acquisitions are used to study the relationship between tissue oxygenation and bioenergetic status (128,129). Tran et al. (130) have shown that it is in principle possible to collect spatial maps of the tissue pO2 on the basis of the Mb signal, using spectroscopic imaging. Such measurements are hampered by the low Mb concentration in muscle (typically 0.2 mM) and the relatively short T2 relaxation time of the proximal histidine proton. For that reason, most studies make use of a surface coil for signal
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localization, without gradient-based volume selection. Since the T1 of the Mb proton is also relatively short, a short repetition time can be chosen to enable an optimal trade-off between signal-to-noise and total acquisition time during dynamic studies. Very recently, Richardson et al. (131) were the rst to assess the local pO2 in resting human skeletal muscle in vivo with 1H MRS, with the use of a 4 T scanner and a large voxel volume. A sufcient signal from deoxy-Mb was obtained to enable the estimation of resting pO2 under normoxic conditions. For further details on MR-based pO2 measurements, the reader is referred to the contribution by Carlier et al. elsewhere in this issue.
13
C MRS
Natural abundance studies. A uniquely strong point of 13C MRS is the very wide chemical shift range of up to 200 ppm, which enables the differentiation of a multitude of chemical species and explains its widespread use in organic chemistry. However, the sensitivity of the 13C nucleus is intrinsically low and its natural abundance is only 1.1%. Therefore, the use of 13C MRS for in vivo studies of skeletal muscle without isotope enrichment is essentially limited to materials that are present at high concentrations, in particular glycogen and triglycerides. Muscle levels of glycogen can be quantied from the intensity of the C-1 peak at 100.5 ppm. The use of 13C MRS to measure muscle glycogen has been pioneered by the Yale group (132136). Jue et al. (132) were the rst to report on the detection of the natural abundance 13C signal from the glycogen C-1 resonance and used this technique to measure glycogen turnover in human muscle (133,134). The glycogen C-1 resonance is 100% visible in spite of its high molecular weight (135), which is due to the high mobility of the a-1,4-glycosidic linkage. Importantly, the comparison with direct assays in needle biopsy samples showed that 13C MRS is capable of accurately measuring muscle glycogen levels in vivo and has a higher precision than the invasive biochemical approach (136). Natural abundance 13C MRS studies have been used to measure the recovery of muscle glycogen after exercise (137,138) as well as glycogen reduction in non-exercising muscle (139). In recent years, the technique has been increasingly used to study aspects of insulin resistance (140) and type 2 diabetes (141). This use of 13C MRS can be expected to increase in the future, considering the steep increase in the incidence of type 2 diabetes that is being seen worldwide. Natural abundance 13 C MRS has also been used to investigate muscle triglyceride content. Perseghin et al. (142) studied the relation between muscle lipid levels and insulin resistance, a major prediction factor for the onset of type 2 diabetes.
Isotope enrichment studies. Most 13C MRS studies involve the use of isotope enrichment of substrates that are introduced into the subject, e.g. via intravenous infusion or ingestion. Although still rather costly, especially for human applications, the use of 13Cenriched compounds opens up unique possibilities. Isotope enrichment has two major advantages. Firstly, it will increase the detection sensitivity, in particular when high fractional enrichments are employed. Secondly, in most cases enrichment at one or two specic positions in the substrate(s) of choice is made use of, which enables specic monitoring of the fate of these carbons. As a consequence, the uxes through specic metabolic pathways can be quantied from time-course 13 C MRS studies. One of the 13C- enriched substrates that is most used in skeletal muscle studies is [1-13C]-glucose, which labels the glycogen C-1 carbon. The Yale group has used this approach to study glycogen synthesis rates in type 2 diabetes patients (143) as well as the mechanism of action of troglitazone (144), a drug which improves insulin sensitivity. Similar studies on insulin resistance and therapeutic interventions, using 13C-enriched glucose infusions and measuring glycogen synthesis with 13C MRS, have been carried out in rat skeletal muscle (145,146). In the latter study, Jucker et al. (146) made use of [1,6-13C2]-glucose. Infusions with 13C-enriched glucose have also been used for very detailed studies of the control of glycogen synthesis and glycolysis, by measuring the rates of glucose label incorporation into [1-13C]- and [6-13C]-glycogen and into [3-13C]-lactate and [3-13C]-alanine respectively (146149). As an example, Fig. 7 depicts the dynamics of 13C label incorporation from [1,6-13C2]-glucose in skeletal muscle of a Zucker fatty rat, a widely used animal model for studies on insulin resistance and type 2 diabetes. Recently, Serlie et al. (150) provided evidence that glycogen synthesis rates in human gastrocnemius muscle as determined from the appearance of 13C label from [1-13C]-glucose at the glycogen C-1 carbon are not representative of whole-body muscle glycogen synthesis. This assumption, which is often employed in studies of glucose metabolism, was proven wrong by comparing the muscle glycogen rates as determined by 13C MRS with the alternative radioisotope method of assessing wholebody glycogen synthesis using [3-3H]-glucose during a hyperinsulinemic clamp. One of the most exciting uses of 13C MRS is the quantication of the ux through the mitochondrial tricarboxylic acid (TCA) cycle, which enables a noninvasive, in vivo assay of the activity of the organelle that is vital for sustained skeletal muscle function. This technique again makes use of the infusion of 13C-enriched compounds, including glucose and acetate. The crux of the technique is that ongoing TCA cycle activity leads to a very characteristic pattern of 13C enrichment in the carbons of glutamate. Although glutamate itself is not an
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intermediate of the TCA cycle, its enrichment pattern nevertheless reports on TCA turnover kinetics because the mitochondrial glutamate pool is in rapid equilibrium with a-ketoglutarate via a transaminase reaction. The 13C label at the C-1 position of glucose will initially enter the C-4 position of glutamate during the rst turn of the TCA cycle and equally label the glutamate C-2 and C-3 positions on subsequent turns through the cycle. Many studies based on this concept have made use of muscle extracts to quantify TCA cycle activity, which has the advantage that detailed isotopomer analysis is feasible with high-resolution MRS. Jucker et al. (147) employed [1-13C]-glucose as the substrate to study the effects of lipid infusions on TCA cycle activity. Mezzarobba et al. (151) used [2-13C]-acetate as the substrate to quantify TCA cycle turnover from the labelling pattern at the C-4 and C-2 positions in glutamate to gain insights into the effects of aging in rat skeletal muscle. It is obviously most attractive to perform the TCA cycle activity tests in vivo and thus maintain the advantage of the non-invasiveness of MR and the possibility of conducting time-course studies in the same subject. Infusions with [2-13C]-acetate have been used to determine the TCA cycle activity as a means of assessing mitochondrial coupling in vivo in rat skeletal muscle (68,69) and human skeletal muscle (71,73). The idea is that TCA cycle ux is a measure of the rate of mitochondrial oxygen consumption by the respiratory chain. By independently measuring the unidirectional ATP synthesis ux with 31P-saturation transfer (see
above), MRS thus enables the in vivo measurement of the P/O ratio, which is a measure of the extent of coupling between mitochondrial ATP synthesis and O2 consumption. These studies in rat muscle showed that coupling was identical in awake fed and fasted animals (69) and diminished upon increased expression of uncoupling protein homolog 3 (UCP3) (68), which has been inferred to play a role in body weight regulation. Similar investigations have also been conducted in skeletal musle of UCP3 knock-out mice (70). Interestingly, 13C MRS studies in human muscle provided evidence of a diminished mitochondrial energy coupling upon treatment with thyroid hormone (71) and during aging (73). The latter ndings led the authors to suggest that an ageassociated decline in mitochondrial function plays a critical role in the development of insulin resistance in the elderly. Marcinek et al. (152,153) have recently proposed an alternative, 31P-MRS- based test to assess mitochondrial functionality in vivo. The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen uxes, P/O) plays a central role in cellular bioenergetics, and reduced P/O values are associated with mitochondrial pathologies. To measure mitochondrial coupling under physiological conditions, Marcinek et al. have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATP utilization rates and oxygen consumption rates during ischemia with 31P MRS (from the rate of PCr depletion) and optical spectroscopy respectively. Applying this
Figure 7. In vivo13C NMR spectra of [1,6-13C2]-glucose label incorporation into [1-13C]- and [6-13C]-glycogen, [3-13C]-lactate and [3-13C]-alanine in skeletal muscle of an awake Zucker fatty rat. A time series of 15 min baseline subtracted spectra acquired during a euglycemic-hyperinsulinemic clamp are shown. [1-13C]-glucose (b-anomer at 96.8 ppm and a-anomer at 93.0 ppm), [1-13C]-glycogen (100.5 ppm), [6-13C]-glycogen (61.4 ppm), [3-13C]-lactate (21.0 ppm) and [3-13C]-alanine (16.9 ppm) are visible where indicated. [Reproduced with permission from (146)]
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approach to mouse skeletal muscle in vivo, the authors found that mitochondria are tightly coupled in vivo (152) and that aging is associated with a gradual decline in coupling efciency (153). A number of studies have made use of the infusion of 13 C-labeled fatty acids. Walton et al. (154,155) have studied the infusion of [2,4,6,8-13C4]-octanoate and analyzed its metabolism with high-eld MRS of postmortem muscle extracts. Very recently, Ravikumar et al. (156) have measured post-prandial fat storage in liver and skeletal muscle of healthy volunteers and type 2 diabetes patients. The authors found that in type 2 diabetics the rate of fatty acid storage is increased in both liver and muscle and propose that this may underlie the elevated tissue triglyceride stores and consequent insulin resistance that is characteristic of this disorder. int Zandt et al. (89) have reported the use of [4-13C]creatine for non-invasive measurement of the ratio of phosphocreatine (PCr) to creatine (Cr) in wild-type and CK-decient mouse skeletal muscle with the use of 13C MRS. To this end, mice were i.v. injected with highly enriched creatine several times. The high chemical shift dispersion of 13C MRS allowed the differentiation of PCr and Cr, thus enabling the straightforward determination of the muscle PCr/Cr ratio. Although lower in CKdecient muscle compared with wild-type tissue, the measured PCr/Cr ratio in the knock-out animals was indicative of a signicant residual CK activity, which was attributed to the B-type CK presumably originating from satelite cells.
MR IMAGING OF SKELETAL MUSCLE

In recent years, a great deal of progress has been made in the use of dynamic 1H MR imaging for measuring the ber architecture, the perfusion and also the mechanical properties and contractile activity of skeletal muscle tissue in vivo. These parameters are closely linked, and their combined measurement in comprehensive scanning protocols yields a wealth of information for the improved understanding of the function of normal muscle and the mechanisms underlying impaired function in muscle disorders.
Diffusion MRI of ber architecture and myocyte integrity

In recent years, diffusion-weighted MRI has matured to become an important tool in basic and clinical biomedical research [for a review, see (157160)]. The technique is based on the measurement of the diffusivity of tissue water. Diffusional displacements of water in isotropic solutions are random (i.e. have equal probability in all spatial directions) and unrestricted. However, water diffusion in biological tissues is often restricted by physical barriers, both in the intra- and in the extracellular
compartment, which causes the effective diffusion coefcient to be lower than that in solution. In addition, diffusional displacements may depend on the direction in which it is probed. This diffusion anisotropy can be exploited to obtain information on the spatial architecture of the tissue. MR is uniquely suited to measure both diffusion restriction and anisotropy effects. Diffusion MR was pioneered by Stejskal and Tanner in the 1960s (161). These authors have introduced the pulsed-eld gradient (PFG) technique which remains the most popular procedure for measuring molecular self-diffusion to date. In PFG sequences, two identical pulsed linear gradients are employed successively to encode and decode the phase of the transverse magnetization of the nuclear spins according to their spatial position along the gradient direction. For static spins, the gradient pulses are selfcompensatory and will cause no net phase effect. However, diffusional displacement along the gradient axis in the time period between the two gradient lobes will leave a net phase effect for individual spins. For an ensemble of spins (that has a Gaussian displacement probability distribution in isotropic solutions), the net phase effect will be eliminated because of the random nature of the diffusion process. However, the magnetization of the spin ensemble will be attenuated to an extent that depends on the diffusion coefcient, the gyromagnetic ratio and a number of experimental parameters that relate to the strength of the diffusion weighting. The latter factor is commonly called the b-value (162). By collecting diffusion-weighted (DW) MR images with at least two different b-values, a parametric image of the apparent diffusion coefcient (ADC) can be calculated. The ADC map collected in this way may depend on many factors, including the diffusion time and, most importantly, the orientation of the tissue relative to the diffusion sensitizing gradient direction. This particularly applies to tissues with an anisotropic organization, of which skeletal muscle is an obvious example. In order to remove this ambiguity, diffusion should be treated as a non-scalar property, which is feasible with the diffusion tensor representation. A minimum of seven images must be acquired in order fully to characterize the diffusion tensor (157), i.e. six images that are weighted for diffusion in six different non-collinear directions and one non-weighted image. This diffusion tensor imaging (DTI) approach yields a number of indices that dene the anisotropic organization of tissue on a pixel-by-pixel level. These indices include the diffusion eigenvalues and the corresponding (orthogonal) eigenvectors, the fractional anisotropy and the ADC. It is important to note that the ADC obtained in this way is a scalar entity that represents the effective diffusivity and no longer depends on the orientation of the tissue relative to the magnet coordinate system. Although DTI of skeletal muscle is still in its infancy, a number of reports suggest that the technique holds great promise for advanced research of muscle architecture. By combined DTI and anatomical studies of rat hindleg
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skeletal muscle, the diffusion tensor eigenvector that corresponds to the highest eigenvalue has been shown to represent the ber direction (163,164). This nding agrees with the intuitive idea that diffusion of water is least hindered along the bers (i.e. parallel to the main elements that can be expected to impose most of the diffusion hindrance and restriction effects, such as the myobrils and the sarcolemma). By denition, the eigenvectors l2 and l3 that correspond to the second and third DTI eigenvalues are oriented perpendicular to the largest eigenvalue l1. It is unclear as yet whether the eigenvectors l2 and l3 have a distinct anatomical basis, i.e. can be assigned to specic features of skeletal muscle n et al. (165) have suggested that, in microstructure. Galba skeletal muscle, l2 is associated with diffusion within the endomysium, and that l3 represents diffusion within the cross-section of the muscle bers. An alternative explanation is that l2 and l3 correspond to the long and short cross-sectional axes of the muscle bers respectively. Irrespective of the anatomical and/or physiological basis of the second and third DTI eigenvectors and eigenvalues, l2 and l3 can be expected to be more sensitive to alterations in the dimensions, the shape and the integrity of the skeletal muscle bers than the principal eigenvalue l1. Indeed, Heemskerk et al. (166) have recently obtained evidence that, among the DTI eigenvalues, l3 shows the largest relative response to
Figure 8. Diffusion tensor eigenvalues (normalized to the control situation) in mouse tibialis anterior (TA) muscle before, during and after 50 min severe ischemic stress. Severe ischemia was imposed on the TA muscle by combined occlusion of the entire hindleg with an inatable cuff and electrical stimulation of the dorsal exor muscles, including the TA, with a chronically implanted electrode, positioned around the common peroneal nerve. The ischemic period is indicated by the cross-hatched area. Histological analysis at 24 h after reperfusion indicated that the TA was seriously damaged. Damage included ber necrosis and inammation. The normal angular appearance of the myocytes was largely lost and cell swelling had occurred. Data are presented as mean SD (n 5),p < 0.05 versus initial value. (Courtesy of A. M. Heemskerk et al., Eindhoven University of Technology)
muscle injury. Figure 8 depicts the time course of l1, l2 and l3 in mouse tibialis anterior muscle before, during and after a period of severe ischemic stress. While the DTI indices remained essentially unchanged during ischemia, l1, l2 and, in particular, l3 showed a large increase (up to 30%) in the hours following reperfusion. Histology at 24 h after reperfusion was indicative of prominent muscle damage. In fact, the DTI indices, especially l3, showed a high correlation with the damage score quantied from the histological slices. These data suggest that DTI may be used as a diagnostic tool to monitor the location and severity of skeletal muscle injury. Future studies will show which of the DTI indices is most informative for the non-invasive monitoring of muscle injury and how this MRI modality compares with the more traditional MRI contrast weightings. Studies on brain tissue have shown that the relationship between the signal intensity on diffusion-weighted images and the b-value increasingly deviates from monoexponential behavior at high b-values (167). Future studies of this kind on skeletal muscle may aid in the differentiation of multiple tissue compartments on the basis of differences in their intrinsic diffusion characteristics. DTI data offer the exciting possibility of reconstructing the muscle ber organization by tracking the ber pathways, using the local, voxel-based principal DTI eigenvalue (168,169). The present authors have recently used this approach to determine the three-dimensional architecture of the mouse tibialis anterior muscle in vivo (170). Figure 9 depicts examples of the possibilities offered by ber tracking in the mouse hindlimb. These data show that ber tracking enables the measurement of ber length, physiological cross-sectional area and pennation angle. Damon et al. (171) recently demonstrated that the pennation angle in rat lateral gastrocnemius muscle as determined by DTI is highly correlated with the value of this parameter as determined by direct anatomical inspection. The above architectural parameters are central to the biomechanical properties of skeletal muscle and hitherto could only reliably be determined by ex vivo anatomical reconstruction methods. The non-invasive MRI-based ber reconstruction opens up the possibility of longitudinal monitoring of alterations in muscle architecture in relation to development, training and disease.
MRI ELASTOGRAPHY AND THE MECHANICAL PROPERTIES OF MUSCLE

It is generally accepted that the elastic properties of tissues, including muscle, are markedly altered by a variety of disease processes (172). The recognition of the potential diagnostic value of the characterization of the mechanical properties of tissues has initiated the search for methods to image tissue elasticity. In materials science, the traditional way of measuring the mechanical
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Figure 9. Example of skeletal muscle ber tracking in mouse hind limb, based on three-dimensional DTI. The gure illustrates the architectural parameters that can be deduced from the data, i.e. pennation angle, ber length, and physiological cross-sectional area (PCSA). In (a) ber tracking was started from the ROIs indicated with the yellow lines. Fibers originating from different ROIs have different colors. The insets in (b), (c) and (d) show the bers projected on a longitudinal MR image for anatomical reference. (a) Transverse slice showing different muscle groups that can be identied in different colors. The image on the right shows an anatomical reference from a different animal. The structures identied using light microscopy were tibialis anterior (TA), extensor digitorum longus (EDL), peroneus (P), gastrocnemius and plantaris (GP), soleus (S), exors (FL) and semimembranosus (Se) muscles and tibia (T) and bula (F). (b) Determination of the pennation angle u, dened as the angle between the tendon sheet (solid line) and the muscle bers (dashed line) in the mid-sagittal plane of the muscle. (c) Tracking of bers in the TA muscle, from which the ber length between the two tendon plates was determined. (d) Longitudinal view of the reconstruction of bers starting from the transversal ROI indicated with the yellow line. The black solid line represents the PCSA, dened as the dot product of the transversal ROI with the local ber direction. [Reproduced with permission from (170)]
properties of a sample is to apply a known stress and to measure the resulting strain. Renements include the use of multiple measurements with variations in the applied stress and/or the use of dynamic rather than static stresses. In recent years, a very elegant MRI procedure has been introduced, that is able non-invasively to measure the elastic properties of tissues, including skeletal muscle. This technique, which has been termed MR elastography (MRE) has been pioneered by Ehman and coworkers (173,174) [for a review, see (172)]. MRE is a technique that can directly visualize and quantitatively measure propagating acoustic strain waves in tissues subjected to harmonic mechanical excitation (173,174). The crux of MRE is that the amplitude and phase of the acoustic waves depend on the mechanical properties of the tissue. Hence, MRE enables the reconstruction of biomechanical parameters such as shear modulus and shear viscosity. MRE scans are performed by synchronizing motionsensitive (i.e. phase-contrast) MR imaging sequences with the application of acoustic waves in the 501000 Hz range. Shear waves in this range of frequencies are useful
because they are much less attenuated than at higher frequencies and their wavelength in tissue and tissue-like materials is in the useful range of tens of millimeters. The MRE sequence is used spatially to map the displacement patterns that are brought about by the mechanical excitation. The harmonic shear waves typically have amplitudes of microns or less. The motion-encoding gradient can obviously be applied along any desired axis to detect cyclic motion in a specic direction. Trigger pulses are used to synchronize an electromechanical actuator, which is coupled to the surface of the object to be imaged, in order to induce shear waves in the underlying tissue at the same frequency as the motionsensitizing gradient. Cyclic motion of the spins in the presence of the motion-sensitizing gradients causes a phase shift in the MR signal. The measured phase shift can be used to calculate the displacement at each imaging voxel, yielding a direct image of the acoustic waves within the object. Spins whose component of motion along the gradient direction are exactly in phase or out of phase with the gradient oscillation have maximum phase
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shifts, with opposite polarity. High sensitivity to smallamplitude motion can be obtained by the use of multiple consecutive cycles of mechanical excitation and motionsensitizing gradient pulses. Most often, two acquisitions are done for each repetition, reversing the polarity of the gradient wave forms. This reduces systematic phase errors and doubles the displacement sensitivity. Obviously, the acquisition of two-dimensional images captures only two of the three components of the wave propagation vector. For that reason, increasing use is being made of three-dimensional sequences (172). By varying the phase offset between the mechanical excitation and the oscillating gradient, the wave patterns can be measured at regularly spaced phase intervals around the cycle. This produces the amplitude as well as the phase of the displacement at each point in space, which are the input to the processing procedures that are used to extract biomechanical properties from the MRE data. In addition, this approach allows the visualization of wave propagation as a CINE loop. MRE has been found to be only sensitive to displacements that are precisely in phase with the sensitizing gradients and is not adversely affected by regular physiological motions (172). Figure 10 shows examples of the use of MRE to measure the propagation of acoustic waves in human skeletal muscle. Several methods can be used to extract mechanical parameters from the MRE-based displacement data. A complete overview is beyond the scope of
Figure 10. MR elastography of human hindleg (A) and shoulder muscle (B). (A) shows a sagittal slice (between the tibia and bula), including (from top to bottom) the tibialis anterior, extensor hallucis longus, soleus, and lateral gastrocnemius. A mechanical driver was placed over the distal end of the gastrocnemius. The signal was sensitized in the vertical direction. The strip holding the driver in place led to the induction of harmonic waves in the tibialis anterior as well. During data acquisition the muscle was relaxed. The FOV was 24 24 cm2 and the data matrix was 256 64 (reconstructed to 256 256), while the shear wave excitation frequency was 100 Hz. (B) shows a planar wave, which originates from the spine of the scapula (on the left side in the image) and propagates in the shoulder muscle towards the medial side (on the right side in the image). The image is raw data, free of ltering and harmonic wave extraction. The FOV was 24 24 cm2 and the data matrix was 256 256, while the shear wave excitation frequency was 150 Hz. (Courtesy of Drs S. I. Ringleb and Q. Chen, Mayo Clinic, Rochester, USA)
this review, and therefore only the basics are described here [for a review, see (172)]. The wavelength of the shear wave is dened as the wave speed divided by the frequency, while the stiffness (or shear modulus) of an incompressible, isotropic and linear elastic material is directly related to wave speed. Thus, for a known input frequency, the estimation of the shear wavelength from the MRE data yields an estimate of the shear modulus. In the simplest case of a single shear wave, this can be done manually. It should be noted, however, that the shear modulus of a material is an absolute quantity that does not depend on the frequency of vibration. Therefore, Ehman and coworkers (175) have introduced the term shear stiffness to indicate the shear modulus of an object at a specic frequency. More elaborate models are currently being developed that are capable of dealing with heterogeneous objects and intrinsically noisy data. Several studies have compared mechanical indices as deduced from MRE with those obtained with classical mechanical testing, mainly on phantom samples (172 174,176178). In general, a good agreement was observed between shear wave MRE and traditional compression tests. Currently, the main clinical interest in MRE arises from the importance of palpation in diagnosis. Palpation assesses the stiffness of a region with respect to surrounding tissue and is the basis for clinical presentation of many breast, thyroid, prostate and abdominal pathologies. MRE provides quantitative stiffness information and enables, in a way, palpation by imaging, even of deep-lying tissues. Several studies have shown the potential of MRE for quantitatively depicting the elastic properties of breast tissue in vivo, and have revealed high shear elasticity in breast tumor tissue and a remarkably large dynamic range to discriminate benign from malignant lesions (179182). The number of biomechanics studies of skeletal muscle with in vivo MRE is still limited. Nevertheless, a number of highly interesting papers have been published. Dresner and colleagues (172,175) have used MRE to assess changes in muscle stiffness with increasing passive loading, using a combination of ex vivo studies on excised bovine muscle and in vivo measurements on human biceps muscle. At each load, eight images were acquired at phase offsets evenly spaced throughout one 150 Hz excitation cycle by altering the phase relationship between the gradient and the mechanical waveforms, as detailed above. The multiple wave images permitted the wave propagation to be visualized. Muscle stiffness, as expressed by the shear modulus, increased linearly for both passive tension (14.5 1.8 kPa/kg) and active tension, in which case the increase in stiffness depended upon muscle size, as expected. Using a largely identical approach, Basford et al. (183) have studied wave-phase propagation in healthy and diseased human tibialis anterior and gastrocnemius muscle. As above, shear-wave wavelength and muscle
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stiffness increased with load in healthy muscle. At rest, the latter two parameters differed signicantly between normal and diseased muscle, suggesting that MRE can have value in assessing neuromuscular disorders. Using a similar study design, Jenkyn et al. (184) have examined several different ankle muscles simultaneously in human subjects using MRE. Applied ankle moment was isometrically resisted at several different foot positions. Shear wavelengths increased in relaxed musle when stretched, and the increase was more signicant with contraction. These ndings agree with the intuition that a stretched muscle will be stiffer than a accid one and that a exed or contracted muscle will be stiffer than a relaxed one. Like the above studies, these ndings suggest that MRE has potential as a non-invasive biomechanical analysis technique that is capable of interrogating any muscle, however deep. Heers et al. (185) have measured the wavelengths of mechanically generated shear waves in the tibialis anterior, gastrocnemius and soleus muscles of healthy volunteers as the subjects resisted ankle plantar-exing and dorsi-exing moments. The MRE elastography wavelengths were compared with electromyography as a measure of muscle activity. The two sets of data showed high linear correlations in all muscle groups examined, suggesting that MRE offers a tool for non-invasive determination of muscle activity. Recently, Uffmann et al. (177) have carried out a careful MRE study in healthy volunteers, in which several extremity muscles at rest were compared. The focus was on the variation in muscle elasticity in a healthy population as well as on the reproducibility of consecutive MRE examinations in follow-up studies. As such, this paper represents a very important reference for future MRE research on diseased and injured muscle. Uffmann et al. (177) found that elasticity values vary substantially, even in healthy subjects, which raises the issue as to what deductions can be reached from MRE scans of patients. The authors offer several suggestions for reducing the variability in the MRE data in future work, including the use of individually optimized loading protocols. In addition, more extensive studies using larger sample sizes should be performed to establish a solid data base of normal biomechanical indices. Along with research in carefully chosen patient populations, studies of this kind are essential for determining the potential of skeletal muscle MRE with regard to clinical use. It is expected that MRE will play a pronounced role in fundamental studies aimed at designing rened models of muscle biomechanics. It is important to note that the analysis of MRE data from skeletal muscle is complicated by the fact that muscle is intrinsically anisotropic in nature. Therefore, the propagation of elastic waves is strongly constrained and directed as compared with the situation in simple isotropic media. Several approaches are being taken to extend the current MRE measurement and analysis
methods to account for ber orientation effects in skeletal muscle [e.g. (186189)].
MRI of muscle perfusion and oxygenation

Although skeletal muscle has a number of nutrient stores that it can use to support muscle activity for a certain time period, prolonged contractile function vitally depends on the adequate supply of oxygen and nutrients and the removal of waste products via the circulation. It is obvious that long-term impairments in the blood perfusion will have deleterious consequences for muscle function. For that reason, considerable efforts have been made in recent years to develop robust MRI procedures for the measurement of the local microvascular perfusion of muscle. Two main MRI techniques from which information on perfusion can be obtained should be distinguished: (a) arterial spin labeling (ASL), which makes use of the dynamic magnetic labeling of owing arterial water to distinguish it from non-owing tissue water (190192); (b) dynamic contrast MRI techniques, which rely on the transient signal change that is caused by the entry of intravenously injected MRI contrast agent in the target tissue (193,194). The detection of the contrast agent can either be based on T1 shortening with T1-weighted MRI (e.g. when Gd-DTPA is used), or T2/ T2 shortening with T2/T2-weighted MRI (e.g. when using blood pool agents based on FeO nanoparticles). In principle, the ASL method is to be preferred, since it makes use of endogenous water to achieve perfusionbased image contrast and does not require the invasive injection of contrast agent. ASL is able to produce quantitative numbers on tissue perfusion by subtracting an image acquired with blood preparation from an image without magnetic labeling of the blood component, combined with modeling using the extended Bloch equations (190192). The fundamental drawback of ASL, however, is that it is relatively insensitive and therefore has considerable difculty in quantifying low perfusion values, such as those occurring in resting skeletal muscle. Closely related to the perfusion methods are MRI procedures that aim to measure changes in the oxygenation status of the blood in response to changes in muscle activity (195198). These techniques are increasingly used to study the balance between muscle activity and the delivery and extraction of oxygen, both in normal and diseased muscle. Recently, Carlier et al. (199) have presented a comprehensive MR protocol for the simultaneous measurement of oxygen supply, uptake and utilization by interleaved 1H MRI and 1H and 31P MRS of the skeletal muscle. Detailed description of the perfusion and oxygenation techniques is beyond the scope of this review. The contribution by Carlier et al. elsewhere in this issue specically deals with the principles and applications of these techniques in skeletal muscle studies.
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T 2 - Weighted MRI of contractile activity

The inherent sensitivity of the relaxation properties of tissue water to its physicochemical environment is one of the main reasons for the remarkable soft tissue contrast in MRI in general and also one of its major strengthts when applied to skeletal muscle. In particular, the T2 relaxation time has been widely used to study water organization and compartmentation in skeletal muscle and how these change with muscle injury (200 202) and contraction [for a review, see (197,203)]. Briey, water T2 increases during muscle activation as a result of osmotically driven intracellular uid shift (197,203). This osmotic driving force is built up intracellularly during ATP metabolism, when a large increase in impermeable molecules occurs (i.e. net breakdown of PCr into Pi and Cr, and, at high workloads, breakdown of glycogen particles into lactate). Early non-MRI studies of T2 relaxation in excised skeletal muscle recognized that the T2 decay curves were multiexponential by nature (204,205). The different T2 components were generally interpreted as arising from different water compartments. In imaging studies of T2 relaxation in skeletal muscle, multiexponential T2 decay has been the subject of only a limited number of studies, mainly because of technical limitations imposed by minimal interecho spacing owing to the need to switch imaging gradients. Mulkern and coworkers have used biexponential echo analysis to study muscle edema, and have provided evidence that the components with fast and slow T2 decay originate from intra- and extracellular water compartments respectively (206,207). The most versatile use of the T2 parameter in studies of skeletal muscle originates from the distinct prolongation of T2 that occurs during muscle contraction. This phenomenon can be readily measured with standard single-echo T2-weighted scans. It is well known that exercise leads to a change in the distribution of water within skeletal muscle, and this shift in water distribution is considered to be at the basis of the above T2 change in exercise (208). The T2 changes correlate well with electromyography (EMG) recordings of muscle activity (209) and increase with increased exercise intensity (210). The prime advantage of the MRI read-out is its ability to produce a three-dimensional view of the entire muscle (or groups of muscles), non-invasively, while the EMG is limited to a supercial recording. Probably, the most important feature of the T2-weighted MRI method is its potential to map spatial variations in activity within a muscle, which enables a unique assessment of the distribution of activated muscle bers during a task (197,211213). Recently, Damon and Gore (214) have modelled the effects of sustained isometric contractions on the T2 relaxation time of skeletal muscle. The authors provided evidence that T2 prolongation at short exercise duration mainly results from blood volume and oxygenCopyright # 2006 John Wiley & Sons, Ltd.
ation changes, as well as the creatine kinase reaction. Effects secondary to glycolysis seem to be the main contributors at later times.
MRI tagging and velocity encoding of muscle contraction

MRI also has a lot to offer to the measurement of the contractile function of skeletal muscle. Ultrafast kinematic MRI procedures may be used dynamically to measure the overall deformation of skeletal muscle complexes during muscle activity. In that case, time series of images are analyzed with the use of anatomic landmarks to distinguish between different muscle groups, for example, within the human upper leg during cycling. These MRI techniques can make fundamental contributions to the movement sciences and the understanding of movement defects. However, they lack the ability to measure the contraction-induced local displacements at any location within the muscle, knowledge of which is vital to the understanding of basic muscle biomechanics and the understanding of local contractile failure. Local displacement data can be used to quantify the distribution of strain within the muscle, which again is highly relevant for understanding its function under healthy and diseased conditions. The distribution of strain in skeletal muscle is still poorly understood, with considerable controversy even about the distribution of strain between tendon and aponeurosis (215). Tagging and phase-contrast sequences are two established MRI methods for tracking local displacements due to motion. Tagging MRI has been pioneered by Zerhouni and Axel and colleagues for the measurement of the local contractile activity of the heart (216219). Much more recently, this MRI technique has been employed to assess the contraction of skeletal muscle. Tagging MRI can be performed in several ways. A frequently used procedure involves the preparation of the longitudinal magnetization with the use of a combination of two RF pulses, separated by a pulsed linear eld gradient, followed by a spoiling gradient for complete dephasing of the transverse magnetization. The result of this preparation sequence is that the longitudinal magnetization exhibits a sinusoidal modulation in the direction of the tagging gradient. The frequency of the signal modulation can be set at will by the choice of the integral of the tagging gradient, which is governed by the level of deformation that is expected: a narrow-spaced pattern can be used for small deformations, while a coarse pattern should be chosen in case of large deformations. The amplitude of the signal modulation depends on the ip angle of the RF pulses. A pair of 908 pulses causes the signal to vary between the thermal equilibrium value and its inverse. In the usual magnitude representation of MR images the signal modulation is seen as periodic bands of signal loss
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(which actually coincide with the zero crossings of the modulation pattern). The above one-dimensional line pattern can be extended to a two-dimensional tagging grid by repeating the signal preparation with a tagging gradient pulse in an orthogonal direction. The crux of tagging MRI is that the tagging pattern is attached to the tissue and therefore muscle activity will result in pattern deformation. This implies that, by dynamically measuring changes in the tagging pattern in successive MRI scans, the temporal phases of muscle activity can be monitored (220,221). Thus, tagging permits direct visualization of tissue motion. The tagging pattern gradually fades owing to T1 relaxation, and therefore tissue deformation can only be measured for a limited time span following pattern application. However, since muscle T1 is typically around 1 s, pattern fading poses no serious limitations to most applications of tagging MRI. Tagging MRI requires rather complex post-processing to compute the displacement of each pixel, in particular when this has to be done in three dimensions. One analysis procedure involves the tracking of the points at which two orthogonal tagging lines intersect as ducial markers of tissue deformation (222). This is a robust procedure but has the disadvantage that it only uses a small fraction of the pixels in the image to track motion. Another procedure that does make use of all pixels is harmonic phase analysis (HARP) (223,224), which is based on the unwrapping of the phase along each row of pixels parallel to the gradient direction (which is fully dened by the sinusoidal modulation) and tracking the tissue displacements pixel-wise from image frame to image frame on the basis of the unwrapped phase. If there is no motion, the phase of the tag pattern remains linear. If there is motion, the phase along the sinusoidal tag pattern
deviates from linearity. The phase change is related to the in-plane displacement of the tissue tags. HARP analysis involves little user intervention and relatively fast processing. Once the displacement data have been obtained, parameters of biomechanical interest can be calculated, including the strain and its temporal derivative, the strain rate. Strain is expressed as the fractional change in length (in percent) from a reference (e.g. resting) state to a state following tissue deformation (e.g. muscle contraction) (225). By convention, contraction or shortening of the muscle bers yields negative strain values, and lengthening results in positive values. Strain maps are typically displayed in color code. It should be noted that strain is a tensor and is characterized not only by the magnitude of the length change of the tissue elements but also by the direction of this change. The description of the strain tensor concept is beyond the scope of this review [see (225)]. An example of the use of tagging MRI to measure deformation of skeletal muscle tissue is shown in Fig. 11. These data were taken from a study in a rat model, in which muscle tissue is subjected to a prolonged period of passive loading to simulate the initiation of pressure sores. Pressure sores are a major healthcare and socioeconomic problem, and surprisingly little is known about their aetiology. Tagging MRI was used to measure the extent of tissue deformation upon passive loading of the muscle, from which the strain was computed pixel by pixel . In this case, the tagging pattern was applied in the non-deformed state of the muscle. This was followed by the rapid application of an indenter to induce muscle deformation, and nally by snapshot MRI read-out. The comparison of the parametric strain image with T2weighted scans (not shown) suggests that muscle injury
Figure 11. Use of tagging MRI to measure rat skeletal muscle deformation due to mechanical loading, as a model of pressure sore initiation. (AC) Transverse images taken before and (DF) after application of a (uid-lled) indenter [visible in (D)]. (A, D) Anatomical images acquired before (A) and during loading (D). (B,C,E,F) Tagging images, in which the tagging grid was applied perpendicular (B,E) and parallel (C,F) to the direction of indentation. The tagging data were used to calculate the level of indentation (G) and the maximum shear strain (H). The dashed lines in (G) and (H) indicate the deformed and undeformed contours of the muscle. (Courtesy of A. Stekelenburg et al., Eindhoven University of Technology)
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Figure 12. Spin tagging MR images showing two-dimensional displacement of different material points in human muscle during isometric contraction. (A) shows the essentially non-deformed tagging pattern as imaged shortly after grid application, while (B) to (D) depict progression of displacement due to contraction. Each contraction cycle corresponded to circa 1400 ms. The time separation between the images as shown is circa 140 ms. The displacement patterns as measured with MRI tagging were used to validate displacement as calculated from phase contrast images (after corrections for phase shading) by superposing the calculated PC-based trajectories on the spin tag lines at each of the cine phases. The trajectories as calculated from the PC images corresponded well with those measured with spin tagging, which can be regarded as almost the gold standard. [Reproduced with permission from (234)]
due to prolonged loading is initiated in high- strain regions, in agreement with the present working hypothesis. An alternative procedure for quantifying regional skeletal muscle function, the phase-contrast (PC) technique, relies on the accumulation of the phase of moving spins proportional to their velocities and to the magnitude of velocity-encoding magnetic eld gradients (226). PC-MRI makes use of bipolar gradient lobes along the desired encoding direction prior to signal acquisition. Since the phase of a pixel is modulated by velocity, functional data can be attained with maximal resolution since each pixel contains velocity data. PC-MRI has been shown to be capable of non-invasive assessment of deformation and motion of skeletal muscle (215,227 230) as well as heart (226,231). PC-MRI enables the computation of trajectories of points within the tissue, from which motion is derived by means of integration of the velocities. Velocity measurements at a given image location as a function of time in general correspond to different material points. Several methods have been described to estimate the trajectory of material points by integrating CINE-PC velocity data along time (232). The motion data in turn enable the pixel-by-pixel calculation of the local strain. It is also possible directly to measure the strain rate (233). Although PC-MRI is intrinsically suited to measuring the velocity in each pixel of the image, signicant errors in quantication are introduced by phase variations arising from sources such as magnetic eld inhomogeneities and eddy currents, in particular when encoding low velocities.
Recently, Sinha et al. (234) have presented a comprehensive protocol for kinematic studies of healthy and atrophied muscle, using a combination of phase contrast and spin tagging techniques. Figure 12 shows that the two techniques yield essentially similar displacement trajectories, when applied to the measurement of isometric contraction of the human lower leg. Recently, yet another technique has been introduced to measure muscle contractile function, i.e. displacement encoding with stimulated echoes (DENSE) (235,236). DENSE modulates the phase of each pixel according to its position rather than its velocity. This is done with the use of a pair of encoding gradients in the two TE/2 periods of a STEAM sequence. The gradient pair is self-compensatory for static tissue, while spins that have moved during the mixing period will retain net phase. Through the use of the stimulated echo technique, relatively long displacement encoding times can be used, which yields improved phase contrast with moderate gradient strength and relatively short echo time. DENSE has been employed to obtain high-resolution phase maps of the heart (235,236). Recently, the rst skeletal muscle study was reported that made use of DENSE to visualize skeletal muscle mechanics during joint motion (237).
CONCLUDING REMARKS
This review has highlighted the opportunities that MR techniques offer to basic biological as well as applied biomedical research on skeletal muscle biomechanics and
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J. J. PROMPERS ET AL. cost transfer function. Proc. Natl Acad. Sci. USA 1985; 82(24): 83848388. Jeneson JA, Westerhoff HV, Brown TR, Van Echteld CJ, Berger R. Quasi-linear relationship between Gibbs free energy of ATP hydrolysis and power output in human forearm muscle. Am. J. Physiol. 1995; 268(6 Pt 1): C14741484. Jeneson JA, van Dobbenburgh JO, van Echteld CJ, Lekkerkerk C, Janssen WJ, Dorland L, Berger R, Brown TR. Experimental design of 31P MRS assessment of human forearm muscle function: restrictions imposed by functional anatomy. Magn. Reson. Med. 1993; 30(5): 634640. Blei ML, Conley KE, Kushmerick MJ. Separate measures of ATP utilization and recovery in human skeletal muscle. J. Physiol. 1993; 465: 203222. Jeneson JA, Wiseman RW, Kushmerick MJ. Non-invasive quantitative 31P MRS assay of mitochondrial function in skeletal muscle in situ. Mol. Cell Biochem. 1997; 174(12): 1722. Crowther GJ, Carey MF, Kemper WF, Conley KE. Control of glycolysis in contracting skeletal muscle. I. Turning it on. Am. J. Physiol. Endocrinol. Metab. 2002; 282(1): E6773. Lanza IR, Befroy DE, Kent-Braun JA. Age-related changes in ATP-producing pathways in human skeletal muscle in vivo. J. Appl. Physiol. 2005; 99(5): 17361744. Meyer RA, Paganinni AT, Stoyanova R, Brown TR. Non-negative least squares (NNLS) analysis of PCr recovery rates in skeletal muscle with mixed ber type. Proc. Meeting ISMRM, 1997; 5: 1309. Connett RJ. Models of steady-state control of skeletal muscle VO2 evaluation using tissue data. Adv. Exp. Med. Biol. 1988; 227: 215219. Gadian DG, Radda GK, Brown TR, Chance EM, Dawson MJ, Wilkie DR. The activity of creatine kinase in frog skeletal muscle studied by saturation transfer nuclear magnetic resonance. Biochem. J. 1981; 194: 215228. Meyer RA. A linear model of muscle respiration explains monoexponential phosphocreatine changes. Am. J. Physiol. 1988; 254(4 Pt 1): C548553. Kushmerick MJ, Meyer RA, Brown TR. Regulation of oxygen consumption in fast- and slow-twitch muscle. Am. J. Physiol. 1992; 263(3 Pt 1): C598606. Harkema SJ, Meyer RA. Effect of acidosis on control of respiration in skeletal muscle. Am. J. Physiol. 1997; 272(2 Pt 1): C491 500. Jeneson JA, Wiseman RW, Westerhoff HV, Kushmerick MJ. The signal transduction function for oxidative phosphorylation is at least second order in ADP. J. Biol. Chem. 1996; 271(45): 27995 27998. Balaban RS. Cardiac energy metabolism homeostasis: role of cytosolic calcium. J. Mol. Cell Cardiol. 2002; 34(10): 1259 1271. Vicini P, Kushmerick MJ. Cellular energetics analysis by a mathematical model of energy balance: estimation of parameters in human skeletal muscle. Am. J. Physiol. Cell Physiol. 2000; 279(1): C213224. Binzoni T, Ferretti G, Schenker K, Cerretelli P. Phosphocreatine hydrolysis by 31P-NMR at the onset of constant-load exercise in humans. J. Appl. Physiol. 1992; 73(4): 16441649. Arnold DL, Matthews PM, Radda GK. Metabolic recovery after exercise and the assessment of mitochondrial function in vivo in human skeletal muscle by means of 31P NMR. Magn. Reson. Med. 1984; 1(3): 307315. Arnold DL, Taylor DJ, Radda GK. Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann. Neurol. 1985; 18(2): 189196. Larson-Meyer DE, Newcomer BR, Hunter GR, Hetherington HP, Weinsier RL. 31P MRS measurement of mitochondrial function in skeletal muscle: reliability, force-level sensitivity and relation to whole body maximal oxygen uptake. NMR Biomed. 2000; 13(1): 1427. Pipinos II, Shepard AD, Anagnostopoulos PV, Katsamouris A, Boska MD. Phosphorus 31 nuclear magnetic resonance spectroscopy suggests a mitochondrial defect in claudicating skeletal muscle. J. Vasc. Surg. 2000; 31(5): 944952. Iotti S, Lodi R, Frassineti C, Zaniol P, Barbiroli B. In vivo assessment of mitochondrial functionality in human gastrocneNMR Biomed. 2006; 19: 927953 DOI: 10.1002/nbm
function. MR-based tools allow the in vivo study of a remarkably broad range of processes ranging from the metabolic activity of the myocyte to the contractile pattern of whole muscle groups. Each of these tools can equally well be used to study animal models and human patients, which makes the transfer of technology from the experimental to the clinical setting a relatively straightforward process. It is expected that the impact of MR technology in skeletal muscle research will continue to grow in the coming years. This is in part inspired by the fact that human disorders, including type 2 diabetes, in which the musculoskeletal system plays a central role, have an increasing socioeconomic impact in modern society.
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Acknowledgements
The Department of Biomedical Engineering of Eindhoven University of Technology generously supported the skeletal muscle research in the authors laboratory. The authors MR research on muscle in animal models has greatly beneted from an investment grant (grant number 91104-018) of the Medical Sciences division of the Netherlands Organization for Scientic Research (NWO).
20. 21.
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NMR IN BIOMEDICINE NMR Biomed. 2006; 19: 927953 Published online in Wiley InterScience (www.interscience.wiley.com) DOI:10.1002/nbm.

Dynamic MRS and MRI of skeletal muscle function and biomechanicsy

Why MRS and MRI?

MR SPECTROSCOPY OF SKELETAL MUSCLE

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

NMR Biomed. 2006; 19: 927953 DOI: 10.1002/nbm

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

MR IMAGING OF SKELETAL MUSCLE

Diffusion MRI of ber architecture and myocyte integrity

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

MRI ELASTOGRAPHY AND THE MECHANICAL PROPERTIES OF MUSCLE

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

MRI of muscle perfusion and oxygenation

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

T 2 - Weighted MRI of contractile activity

MRI tagging and velocity encoding of muscle contraction

SKELETAL MUSCLE BIOMECHANICS AND FUNCTION

15. 16. 17. 18. 19.

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35. 36. 37. 38.

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Copyright # 2006 John Wiley & Sons, Ltd.

NMR Biomed. 2006; 19: 927953 DOI: 10.1002/nbm

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